吉玛慢病毒再次助力我国优秀科学家登顶国际顶级期刊《CELL》

2020年9月14日，中山大学孙逸仙纪念医院 苏士成博士、许小丁博士及中山大学附属第三医院 高志良教授作为共同通讯作者，在国际顶尖学术期刊  Cell 在线发表题为：Targeting Mitochondria-Located circRNA SCAR Alleviates NASH via Reducing mROS Output 的研究论文。该研究确定了一种 线粒体特异性circRNA——SCAR，在 非酒精性脂肪性肝炎（NASH）中表达下调，向线粒体特异性递送SCAR，可降低线粒体活性氧 （mROS）输出，减轻炎症，可作为NASH的治疗靶标。

非酒精性脂肪性肝炎（NASH）是 非酒精性脂肪性肝病（NAFLD）的炎症亚型，伴有肝脂肪变性以及肝细胞损伤和炎症的证据，伴或不伴肝纤维化。随着时间的推移， NASH可能进展为肝硬化、终末期肝病或需要肝移植。非酒精性脂肪性肝炎患者的肝病致残率和病死率显著高于单纯性脂肪肝。

线粒体（mitochondrion），是细胞的“ 能量工厂”，线粒体发挥重要的细胞功能，通过氧化磷酸化 （OXPHOS）调节能量供应，执行细胞死亡以及Ca离子和活性氧（ROS）的细胞内信号转导，线粒体 在免疫代谢疾病中也起到了关键作用。

研究团队通过对非酒精性脂肪性肝炎（NASH）患者肝成纤维细胞的circRNA表达谱分析，发现线粒体circRNA占NASH成纤维细胞下调circRNA的相当一部分。观察到位于线粒体中的 circRNA——SCAR，抑制线粒体活性氧 （mROS）输出和成纤维细胞活化。由PGC-1α介导的circRNA SCAR与ATP5B结合，并通过构建慢病毒shRNA干扰阻断CypD-mPTP相互作用而关闭 线粒体通透转运孔道（mPTP）。脂质超载通过内质网应激诱导的 C/EBP-同源蛋白（CHOP）抑制PGC-1α。动物体内实验也表明，靶向circRNA SCAR可减轻高脂饮食引起的肝硬化和胰岛素抵抗。而在临床上，circRNA SCAR与脂肪变性到NASH的进展有关。

通过该研究，我国科学家又进步确定了一种线粒体中的新型circRNA——SCAR，可作为非酒精性脂肪性肝炎（NASH）的治疗靶标，为我们今后攻克该疾病提供了新的研究方向。

 
 


Figure 5. circRNA SCAR Shuts Down mPTP by Preventing CypD-mPTP Interaction

(A) Normal fibroblasts were treated with palmitate in the presence of mito-NP containing empty vectors (Vector), wild-type (SCAR), or mutated (SCAR-mut) circRNA SCAR overexpression vectors. Their mPTP opening was measured by calcein loading/CoCl2 quenching (n = 4 patients).

(B) In vitro RNA/protein interaction assay between recombinant ATP5B and indicated biotin-labeled circRNAs (n = 3).

(C) Normal fibroblasts were treated with palmitate in the presence of mito-NP containing wild-type or mutated circRNA SCAR overexpression vectors. Representative western blots of CypD and ATP5B (subunit of ATP synthase) in the ATP synthase-immunoprecipitated lysates are shown (n = 3 patients).

(D–G) Normal fibroblasts were treated with palmitate in the presence of mito-NP containing wild-type or mutated circRNA SCAR overexpression vectors. In some experiments, fibroblasts were pre-transduced with CypD-shRNAs.

(D) mPTP opening (upper) and ΔΨm (lower) were measured by calcein loading/CoCl2 quenching and JC-1 staining, respectively (n = 3 patients).

(E) Cytosolic ROS levels were detected by cytoORP via confocal microscopy. The 405/488 nm excitation ratio was quantitated (n = 5 patients).

(F) Fibroblast contractility in collagen matrices (n = 3 patients).

(G) Indicated cytokines were examined by ELISA (n = 4 patients).

注：材料方法中shRNA and siRNA Mediated Silencing

For shRNA transduction, a lentiviral vector plasmid pLKO.1-puro (GenePharma Inc, Shanghai, China) was used to construct the stable clones. 

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参考文献

1. Zhao et al., Targeting Mitochondria-Located circRNA SCAR Alleviates NASH via Reducing mROS Output, Cell (2020), https://doi.org/10.1016/j.cell.2020.08.009
2. C.Y. Yu et al.,The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency. Nat. Commun., 8 (2017), p. 1149